Through metabolic engineering of the yeast glycosylation pathway, GlycoFi is developing yeast capable of secreting recombinant proteins with human-like glycosylation. In Phase I, a toolkit of cloning vectors, strains, libraries and protocols were developed for targeting glycosylation enzymes to the yeast secretory pathway. The feasibility of this approach was demonstrated by the functional expression of sigma-1,2,-mannosidase in vivo, resulting in the production of a recombinant protein with 65 percent human-like Man5glycosylation structure. This Phase II proposal aims to develop an automated, high-throughput approach capable of screening 3000 samples per week for highly efficient enzyme catalytic domains. These enzymes will be incorporated into a production strain generating commercially important proteins with a human-like Man5glycosylation structure. Proposed Commercial Application: A shortage of manufacturing capacity has emerged as the single largest barrier preventing therapeutic proteins in the development pipeline from reaching the clinic. GlycoFi's novel yeast-based protein production platform will alleviate this bottleneck and allow the bio-pharmaceutical industry to bring their products to market in a faster and more cost effective manner.